Our findings are in line with the view that, in physiological conditions, p110γ activity undergoes a delicate negative ZD4054 186497-07-4 regulation in response to cAMP production and PKA activation. This inhibitory effect can be linked to the well-established view that, while class IA exerts beneficial effects on the myocardium , p110γ function is associated with detrimental responses to cardiac stress. In heart failure, p110γ is upregulated, and due to defective PKA-mediated inhibition, its activity is significantly enhanced. Of note, PtdIns P3 measurement in TAC-treated hearts showed that only in p110γKD/KD and not in p110βKD/KD hearts is PtdIns P3 lower than in wild-type controls, thus confirming a prominent role of the p110γ isoform in heart failure. Perino et al. Page 7 Mol Cell.
AEE788 EGFR inhibitor Author manuscript; available in PMC 2012 January 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript The negative influence exerted by p110γ catalytic activity on the development of heart failure appears to be related to its impact on β-AR pathway, a key regulator of heart contractility. Indeed, p110γ promotes the desensitization and downregulation of β-ARs through its interaction with β-ARK1 and through the recruitment of PH domain-containing proteins such as AP-2 , required for the assembly of β-AR downregulation machinery. Consistent with these observations, genetic ablation of p110γ, expression of a catalytically inactive p110γ, or administration of a selective p110γ inhibitor to wild-type mice slightly but significantly increased cardiac β-AR density.
A trend toward elevated β-AR surface expression has been already reported in a previous study conducted on p110γ-deficient mice. In our hands, while basal myocardial β-AR expression was only marginally affected by the inactivation of p110γ, the effect of p110γ on β-AR downregulation appeared prominent during adrenergic stress and pressure overload-induced heart failure. Consistently, in failing hearts, p110γ catalytic activity appeared significantly enhanced and occurred in a context where expression of p110γ and its adaptor p101 was dramatically upregulated. This effect limited the organization of complexes with p84/87 and PKA, thus reducing PKA-mediated inactivation of p110γ. In agreement, blockade of p110γ activity either genetically or pharmacologically led to a renormalization of β-AR density in heart failure, improving compromised cardiac contractility.
In summary, our results establish that myocardial p110γ connects the PtdIns P3 and cAMP signaling pathways. We show that anchored PKA is the key regulator of enzymes in this macromolecular complex and that PKA locally controls PDE3B activity, reducing cAMP levels. This finding provides an explanation for the longstanding conundrum of how the p110γ kinase-independent function can promote cAMP degradation. On the other hand, PKA inhibits p110γ activity to maintain myocardial β-ARs on the cell surface. In heart failure, uncoupling of p110γ from its negative regulator PKA results inβ-AR downregulation. Pharmacological inhibition of p110γ restores the physiological condition, with beneficial effects on β-AR density and, ultimately, on cardiac contractility, thus establishing p110γ targeting as a potential treatment for heart failure.
EXPERIMENTAL PROCEDURES Mice p110γ knockout , p110γ kinase-dead , and p110βkinase-dead mice are all in a C57BL/6J background. C57BL/6J wildtype mice were used as controls. Hearts and Cell Lysis, Protein Immunoprecipitation, and Western Blotting Hearts, adult rat cardiomyocytes, and HEK293T cells were homogenized in 1% Triton X-100 buffer with protease and phosphatase inhibitors. Lysates were cleared by ce
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